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Dynamic Biochemistry, Process Biotechnology and Molecular Biology ©2012 Global Science Books

Optimization of Pretreatment of Wheat Straw

Using Alkali and Biphasic Acid Hydrolysis
Sravanthi Koti • Sai Prashanti • Jahnavi Gentela • Srilekha Kothagauni •
Sridevi Jagavati • Venkateswar Rao Linga*

Department of Microbiology, Osmania University, Hyderabad-500 007, India

Corresponding author: * vrlinga@gmail.com

ABSTRACT
Pretreatment is an important step in the conversion of biomass to biofuels. Alkali pretreatment increases cellulose digestibility and it is the
most effective method for lignin solubilization, exhibiting minor cellulose and hemicellulose solubilization compared to acid or
hydrothermal processes. The present study describes the potential of pretreatment of wheat straw to degrade lignin and facilitate
conversion of polymers to single monomers using alkali treatment followed by biphasic acid hydrolysis. Two alkalis (NaOH and KOH) at
various concentrations (0.25, 0.5, 0.75 and 1 M) and at two incubation periods (6 and 12 h at room temperature) were used to delignify
wheat straw. 1 M NaOH incubated for 6 h significantly degraded lignin (77%) with minimum sugar loss (1.6%). Further, delignified
wheat straw was subjected to two different biphasic acid hydrolyses; firstly, biphasic acid hydrolysis was performed with 2% sulphuric
acid at 121°C for 1 h followed by 4% sulphuric acid at 121°C for 1 h. Secondly, biphasic acid hydrolysis was performed with 3%
sulphuric acid followed by 4% sulphuric acid under the same conditions. The conversion of holocellulose (cellulose + hemicellulose)
during the above two different biphasic acid hydrolyses was 52 ± 2 and 41 ± 2%, respectively.
_____________________________________________________________________________________________________________

Keywords: alkali-lignin
Abbreviations: ADL, acid detergent lignin; KOH, potassium hydroxide; NaOH, sodium hydroxide; NDF, neutral detergent fibre

INTRODUCTION
Worldwide attention has recently turned to bioethanol pro-
duction as a strategy to combat global warming and to im-
prove global energy security. However, feedstocks of cur-
rent bioethanol production methods are currently derived
from edible parts of food crops such as sugarcane, rice
straw, wheat straw and corn (Sakai et al. 2007). Ligno-
cellulosic materials are abundant and cheap feed stock, but
the processing techniques required for ethanol production
are expensive. The cost of ethanol produced from lingo-
cellulosic materials with currently available technology
under the present economic conditions is not competitive
with the cost of gasoline. Comprehensive process develop-
ment and optimization are still required to make the process
economically viable.
Wheat straw is annually generated in abundance world-
wide (529 million tons/year) (Kim and Dale 2004). This
huge amount of residues may constitute a promising raw
material that could potentially be transformed into a more
edible feed for ruminants or alternatively it could also be
used for the production of ethanol (Rodrigues et al. 2008).
The cell wall of wheat straw consists mostly of cel-
lulose fibres. Cellulose is a linear crystalline polymer of (1-
4)--D-glucose. Hemicellulose is an amorphous and partly
crystalline polymer, which mostly consists of (1-4)--
xylose. Lignin, a phenolic polymer, binds the fibres together.
Hemicellulose and lignin protects the cellulose and this
leads to pretreatment of wheat straw before the hydrolysis.
It binds to side chains of xylans of cell wall and it hinders
the microbial digestion of carbohydrates (Antongiovanni et
al. 1991). Depending on the composition of guaiacyl (G),
syringyl (S) and p-hydroxyphenylpropane (H) units, wheat
straw lignin has been justified as GSH-lignins (Gramineae
lignins from grasses), which are known to be different from
those of softwood (G-lignins), hardwood (GS-lignins) and
compression wood (GH-lignins) lignins. Moreover, phe-
nolic acids, mainly p-coumaric and ferulic acids have been
suggested to form cross-links between lignins and hemi-
celluloses, thus participating in the reticulation of the wheat
straw cell walls (Billa and Monties 1995).
In the present study wheat straw was used as the ligno-
cellulosic substrate since it represents an abundant renew-
able energy source for bioconversion processes as well as
raw material for the production of ethanol (Fang et al.
2002). Pretreatment of the biomass is required to hydrolyze
the hemicellulose and make the cellulose more accessible.
An efficient pretreatment system is crucial to the enzymatic
or acid hydrolysis and thus the fermentation process, which
are the essential steps in the cellulose conversion process to
produce bioethanol (Mosier et al. 2005). The present study
consists of two separate parts, alkali pretreatment and bi-
phasic acid hydrolysis of wheat straw. Alkali extraction
causes solubilization of lignin and modifications in the
crystalline state of the cellulose (Carvalheiro et al. 2008).
These effects can lower or counteract the positive effects of
lignin removal (Gregg and Saddler 1996). According to
Taherzadeh et al. (2008), NaOH causes swelling, increasing
the internal surface of cellulose and decreasing the degree
of polymerization and crystallinity, which provokes lignin
structure disruption. Biphasic hydrolysis studies were con-
ducted to separately hydrolyze hemicellulose and cellulose
fractions present in the wheat straw (Lee et al. 2009). Dilute
acid hydrolysis was done in the first step as it can achieve
high reaction rates and significantly improve cellulose
hydrolysis (Esteghlalian et al. 1997). In the second step,
concentration of acid was increased to get possibly the re-
maining sugars. Moderate temperature (121°C) was applied
in both the steps of acid hydrolysis since higher tempera-
tures in dilute acid hydrolysis cause degradation of mono-
saccharides and formation of inhibitor compounds like
furans, weak carboxylic acids and phenolics which ulti-
mately inhibit the fermentation process (Olsson and Hahn-
Hagerdal 1996; Larsson et al. 1999; Klinke et al. 2004).

Received: 29 March, 2011. Accepted: 28 December, 2011.

Original Research Paper
 Dynamic Biochemistry, Process Biotechnology and Molecular Biology 6 (Special Issue 1), 91-94 ©2012 Global Science Books

MATERIALS AND METHODS Alkali pre-treatment

Raw material Alkali pretreatment showed effective lignin solubilization

Wheat straw was collected from Aligarh, Uttar Pradesh, India. The
straw was dried and processed in a laboratory pulverizer to attain a
particle size between 1 and 3 mm followed by sieving. Then it was
washed with running tap water to make it clear and dust free and
then oven dried at 60 ± 0.5°C.
Analysis of chemical composition of wheat straw
The cellulose, lignin and hemicellulose fractions of pulverized
wheat straw were determined according to the Van Soest et al.
(1991) method. Wheat straw was grinded in a pulverizer through a
1 mm screen and then dried at 50°C for 24 h. The straw was then
analyzed in triplicate for neutral detergent fibre (NDF) and acid
detergent lignin (ADL) using the procedure of Van Soest et al.
(1991). Hemicellulose was calculated as a difference between
NDF and ADL. Cellulose, lignin and ash were determined ac-
cording to the above method.
Alkali pre-treatment
The alkali pretreatment method was carried out by taking 50 g
chopped and dried wheat straw at 10% (w/v) in 500 ml of alkali
(KOH and NaOH) of various concentrations (0.25, 0.5, 0.75 and 1
M) at room temperature (30 ± 2°C) for two different incubation
periods (6 and 12 h). The delignified filtrate was pressed through
cheese cloth and the amount of reducing sugars and phenolics in
the filtrate were measured by using DNS (Miller 1959) and Folin
Ciocalteau’s method (Singleton et al. 1965), respectively. The resi-
dual substrates were washed and neutralized with water and then
oven dried at 50°C for further acid hydrolysis.
Biphasic acid hydrolysis
exhibiting minor cellulose and hemicellulose solubilization.
Among the two alkali treatments (NaOH and KOH), treat-
ment with NaOH showed better delignification. In all the
pretreatment experiments with increasing concentrations of
alkali, the recovery of lignin increased slightly. Comparing
the treatment of wheat straw with NaOH and KOH at vari-
ous concentrations (0.25, 0.5, 0.75 and 1 M), a higher lignin
solubilization performance was observed when the wheat
straw was treated with 0.75 M NaOH and 1 M NaOH at 6 h
(76% ,77%) (Fig. 1) and 12 h (77%, 79%) (Fig. 2) of incu-
bation. Prolonging pretreatment time (up to 12 h) at the
same concentration led to a little increase in lignin deg-
radation. Cheng (1993) reported 60-70% of lignin removal
from wheat straw by using 1.5% NaOH at 100°C for 1 h.
Though the sugar loss is slightly higher, it is < 5%. Free
phenolic monomers in each of the alkaline treatment hydro-
lyzates were isolated and analyzed. Though the percentage
of delignification was slightly higher at 12 h of incubation,
the difference is insignificant.
NaOH loading had a very significant influence on the
degree of delignification. Lignin degradation dramatically
increased with NaOH loading. These findings imply that
higher NaOH loading can remove more lignin and also dis-
solve fewer carbohydrates. According to these experimental
results, 1M NaOH loading at room temperature for 6 h was
optimum.
Biphasic acid hydrolysis
The best alkali pretreated residual substrate was subjected
to two different biphasic acid hydrolyses. Concentrated acid
such as H2SO4 was used to treat delignified wheat straw as

The KOH and NaOH pretreated wheat straw was subjected to two 90
different biphasic acid hydrolyses at 10% (w/v) substrate. First bi- 80
phasic acid hydrolysis was done with 2% sulphuric acid at 121°C 70
for 1 h followed by 4% sulphuric acid at 121°C for 1 h. The sec-
Delignification (%)

60
ond biphasic acid hydrolysis was performed with 3% sulphuric
acid followed by 4% sulphuric acid using the same conditions. 50

The hydrolysate obtained was analyzed for the amount of reducing 40

sugars and phenolics present in it. 30

20
Analytical methods
10

Total reducing sugars present in delignified wheat straw filtrate 0

and acid hydrolysate were estimated by the dinitrosalicylic acid
0

0
H

H
H

H
aO

aO

aO

aO

O
KO

KO
5K

1K

method (Miller 1959). The phenolics were estimated by Folin Cio-

5N

1N
N

N
25

75
0.
25

75
0.

0.

0.

calteau’s method (Singleton et al. 1965).

0.

0.

Concentration of alkali

Statistical analysis Fig. 1 Delignification of wheat straw using different concentrations of

NaOH and KOH at 6 h.
To assess whether there was any significant difference between the
concentration of alkali and percentage of delignification and bet-
90
ween concentration of acid and concentration of sugars obtained, a
correlation test and two paired sample t-tests were performed 80
using SPSS (software for windows release, 17.0, SPSS Inc., Chi- 70
Delignification (%)

cago, IL, USA). 60

50
RESULTS AND DISCUSSION
40

Analysis of chemical composition of wheat straw 30

20
The wheat straw samples were analysed for chemical com- 10
position in triplicates (Van Soest et al. 1991). The average
0
percentages of moisture, lignin, cellulose, and hemicel-
lulose were found to be 8.3, 19.5, 33.5 and 25.5%, respec-
0

0
H

H
H

aO

O
aO

aO

aO

O
KO
KO

5K

1K
5N

1N
N

tively. The percentages of holocellulose and ash were 59

25

75
0.
25

75
0.

0.

0.
0.

0.

and 10.8%, respectively.

Concentration of alkali

Fig. 2 Delignification of wheat straw using different concentrations of

NaOH and KOH at 12 h.

92
 Study on pretreatment and biphasic acid hydrolysis of wheat straw. Koti et al.

25 Treatments (left to right)

Concentration of sugars (mg/ml)

2% 0.25M NaOH
20
2%-4% 0.25M NaOH
15 2% 0.5M NaOH

2%-4% 0.5M NaOH

10
2% 0.75M NaOH
5
2%-4% 0.75M NaOH

0 2% 1M NaOH

Acid concentration (%) 2%-4% 1M NaOH

Fig. 3 Sugars released during biphasic acid hydrolysis (2% followed by 4%).

Treatments (left to right)

18
3% 0.25M NaOH
Concentration of sugars (mg/ml)

16
14 3%-4% 0.25M NaOH
12 3% 0.5M NaOH
10
3%-4% 0.5M NaOH
8
6 3% 0.75M NaOH
4
3%-4% 0.75M NaOH
2
3% 1M NaOH
0

Acid concentration (%) 3%-4% 1M NaOH

Fig. 4 Sugars released during biphasic acid hydrolysis (3% followed by 4%).

Table 1 Sugars present in biphasic acid hydrolysate (2% followed by 4%). Table 2 Sugars present in biphasic acid hydrolysate (3% followed by 4%).
Alkali pretreated 2%+4%AH Sugar 2%+4% AH Alkali pretreated 3%+4%AH Sugar 3%+4% AH
substrate concentration Sachharification substrate concentration Sachharification
(mg/ml) (mg/ml)
0.25 M NaOH 23.90 29 0.25 M NaOH 16.80 21
0.5 M NaOH 23.60 29 0.5 M NaOH 19.33 24
0.75 M NaOH 22.40 27 0.75 M NaOH 12.40 13
1 M NaOH 35.60 52 1 M NaOH 33.23 41

it is a powerful agent for cellulose hydrolysis. In addition, gle phase. Besides sugars, the hydrolyzate contained dif-
concentrated acid must be recovered after hydrolysis to ferent and varying amounts of toxic components like fur-
make the process economically feasible (von Sivers and fural, soluble lignin derivatives which are known to inhibit
Zacchi 1995). microorganisms to produce ethanol.
Figs. 3 and 4 show the yields of sugars produced by 2 The total yield of sugars in first biphasic acid hydrolysis
and 3% sulphuric acid hydrolysis of wheat straw delignified i.e., 2% acid hydrolysis followed by 4% acid hydrolysis
by various concentrations of NaOH (since NaOH showed was 35.4 gL-1 (52%) (Table 1) and the sugar yield in the
maximum delignification, NaOH treated wheat straw was second biphasic hydrolysis was 27.9 gL-1 (41%) (Table 2).
used further in acid hydrolysis studies). Dilute sulphuric
acid at moderate temperature (60-110°C) effectively re- Statistical evaluation of delignification and
moves and recovers most of the hemicellulose as dissolved biphasic acid hydrolysis
sugars (Lu et al. 2008). The present results also indicate that
dilute sulphuric acid treatment (2 and 3%, v/v) effectively To assess whether there was any significant correlation
released a maximum amount of sugars from wheat straw. between the percentage delignification and concentration of
The reducing sugar yields of delignified wheat straw with 1 alkali, a correlation test was performed (Table 3). As there
M NaOH in the first step i.e., 2 and 3% of the two biphasic was a positive correlation (Pearson correlation = 0.582)
hydrolysis methods were 19.6 gL-1 (24.1%) and 17 gL-1 between the percentage of delignification and concentration
(20.9%), respectively. Khokhar et al. (2010) reported 17.71 of alkali at 95% confidence interval (sig: 0.018, P < 0.05),
gL-1 yield after 2% sulphuric acid hydrolysis at 121°C for 1 the percentage of delignification increased with an increase
h. The sugar yield in the second step i.e., 4% sulphuric acid in the concentration of alkali. Table 4 shows the paired t-
hydrolysis in both biphasic acid hydrolysis methods were test which was performed to assess differences between the
15.93 gL-1 (23.6%) and 16.23 gL-1 (22.9%), respectively mean values of percentage of delignification by NaOH and
(Figs. 3, 4). Schmidt et al. (1998) reported 8.8 gL-1 in 4% KOH at 6 and 12 h. Since there was no significant dif-
sulphuric acid hydrolysis at 121°C for 1 h although in a sin- ference between 6 and 12 h of treatment with both NaOH

Table 3 Statistical evaluation (Pearson’s correlation) of concentration of alkali and percentage delignification.
Mean Std. Deviation N Concentration of alkali Percentage of delignification
concentration of alkali 0.6250 0.28868 16 Pearson Correlation 1 0.582*
percentage of delignification 67.6031 7.57442 16 Pearson Correlation 0.582 1
* Correlation is significant at the 0.05 level (2-tailed). n = 16

93
 Dynamic Biochemistry, Process Biotechnology and Molecular Biology 6 (Special Issue 1), 91-94 ©2012 Global Science Books
Table 4 Statistical evaluation (paired samples test) of percentage delignification by NaOH and KOH after 6 and 12 h.
Mean Mean
difference Deviation Mean
Pair 1 percentage delignification by NaOH after 6 h 73.1232 -.40619
percentage delignification by NaOH after 12 h 73.5294
Pair 2 percentage delignification by KOH after 6 h 62.5655 1.68186
percentage delignification by KOH after 12 h 60.8836
Table 5 Statistical evaluation (paired samples test) of concentration of sugars by biphasic acid hydrolsis (2% acid followed by 4% and 3 % acid followed
by 4%).
Mean Mean Std.
difference Deviation Mean
concentration of sugars 2+4 13.2082 3.00518 2.71614
concentration of sugars 3+4 10.2030
** Mean difference is significant at the 0.01 level (2-tailed).
(sig: 0.877, P > 0.05) and KOH (sig: 0.509, P > 0.05), a 6-h
incubation period was selected for further studies.
Another paired t-test was performed (Table 5) to deter-
mine the difference between the mean values of concentra-
tion of sugars and the percentage of acid used in biphasic
acid hydrolysis using sulphuric acid (2% acid followed by 4
and 3% acid followed by 4% sulphuric acid hydrolysis).
Since there was a significant difference between the means
of concentration of sugars in 2% acid hydrolysis followed
by 4 and 3% followed by 4% at a 99% confidence interval
(sig-0.000 P > 0.01), 2% followed by 4% acid hydrolysis
was found to be the best method for the maximum sac-
charification of wheat straw.
CONCLUSION
Pretreatment is a necessary element in bioconversion of lig-
nocellulosics to fuels and other chemicals. Although various
chemical methods of pretreatment for cellulosic materials
have been proposed, their effectiveness varies, depending
on the substrate. Hence, a suitable pretreatment must be
optimized for each substrate. In the present study optimiza-
tion and comparison of different variables for pretreatment
and acid hydrolysis have been carried out successfully. A
maximum of 77% delignification was achieved using 1 M
NaOH at room temperature for 6 h. The highest sugar con-
centration of 35.4 gL-1 representing 52% of holocellulose
was released by hydrolyzing the substrate at 121°C for 1 h
in two phases i.e., 2% followed by 4% sulphuric acid. Ins-
tead of harsher conditions, mild reaction temperatures were
applied during delignification and acid hydrolysis to avoid
the degradation of sugars.
ACKNOWLEDGEMENTS
The present work has been supported by Department of Biotech-
nology (DBT ISLARE), Ministry of Science and Technology,
(Government of India). The authors thank Dr. Smitha Hasini
Panda (Research Associate, CSIR, Department of Microbiology,
Osmania University) for her suggestions and Swarupa Rani (Re-
search Scholar, Department of Microbiology, Osmania University)
for kind support in statistical analysis. The authors also thank Dr.
Jaime A. Teixeira da Silva for significant improvements to gram-
mar and style.
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